RNA interference is a new and powerful research tool that can be used to down regulate or knock out a specific gene or genes in a variety of cell types and organisms. Currently this technology is being used to study gene function, but may one day be used for human therapeutics. The RNAi phenomenon was only recently demonstrated to be present in mammalian cells and in mammalian cells occur specifically in response to short double stranded RNA molecules termed small interfering RNAs (siRNA). In our research, we have discovered that siRNA molecules targeted to different regions of a mRNA vary strikingly in their ability to down regulate the expression of the target gene. Since the mechanism of action by which siRNA acts is speculative, we do not know the reason for this variability in potency. In order to be able to reliably design potent siRNA using something other than an expensive hit-or-miss approach, the mechanism behind the phenomenon and the guidelines of siRNA design must be determined. We propose to perform a detailed and systematic analysis to elucidate guidelines of effective siRNA design. As part of this research, we will test our guidelines by producing siRNA molecules to a panel of genes that have academic and clinical importance, each of which function in a well-defined biological pathway. Finally, we will investigate methods to make siRNAs and methods and reagents to make siRNA transfection more efficient. Several products will be generated from the research performed under this SBIR. First, the guidelines for siRNA design will be used to design highly active gene specific siRNAs that will be sold as controls, functional studies, therapeutics or diagnostics. We will also sell kits that will be used for the enzymatic production of siRNAs. This will reduce the cost of siRNA synthesis significantly. Lastly, we will sell transfection agents that allow for siRNA uptake and activity in mammalian cells.